A novel bA3/A1-crystallin gene mutation results in persistent fetal vasculature

描述(由申请人提供)：此申请是对最近宣布的可用于竞争性修订申请的恢复法案资金的回应(非-OD-09-058)。它将在我们的实验室创造两个新的工作岗位，并帮助保留约翰霍普金斯大学医学院ES细胞靶向核心实验室的几个工作岗位，因为这份修订后的申请中提议的一些研究将在核心设施作为付费服务完成。我们之前描述了一种自然发生的突变(Nuc1)，该突变发生在Spraogue-Dawley大鼠身上，具有一种新的眼部表型，涉及白内障、胎儿血管保留和视网膜发育异常。我们最近报道了晶状体和视网膜发育异常的Nuc1表型是由？A3/A1-晶体蛋白基因突变引起的。？A3/A1-晶体蛋白在晶状体纤维细胞中表达；我们最近的研究表明，在神经视网膜中，它只在星形胶质细胞中表达。持续性胎儿血管(PFV)是一种人类疾病，是由于胎儿血管系统未能退化而引起的。这是一种常见的先天性眼睛发育障碍，在其他正常的儿童中发现。PFV病的根本原因尚不清楚。Nuc1自发突变大鼠是唯一准确模拟人类PFV病所有临床症状的模型。在目前的竞争性补充中，我们建议进一步鉴定我们最近建立的？A3/A1-晶体蛋白转基因小鼠系，并产生条件基因敲除小鼠，其中？A3/A1-晶体蛋白将选择性地在星形胶质细胞或晶状体中缺失。这些基因工程小鼠品系将为功能获得和功能丧失的研究提供额外的工具，并将产生更明确的数据，说明消除或调节？A3/A1-晶状体蛋白在PFV疾病病因学中的作用。本修订申请中提议的研究是基于在父母赠款下迄今取得的进展，并专门设计以加强该赠款的具体目标中概述的实验。我们的工作假设是“A3/A1-晶体蛋白的突变导致星形胶质细胞与玻璃体动脉的异常联系，从而抑制胎儿血管系统的退化”。为了验证这一假设，在父母的资助中提出了以下具体目标：特定目的1：表征和比较A3/A1-晶体蛋白在野生型和Nuc1纯合子大鼠晶状体纤维细胞和星形胶质细胞发育过程中的表达。特异性目的2：探讨Nuc1纯合子星形胶质细胞在发育过程中运动性改变是否导致星形胶质细胞与玻璃体血管系统的异常联系。特异性目的3：探讨表达突变型A3/A1-晶状体蛋白的星形胶质细胞产生的血管内皮生长因子是否参与了Nuc1大鼠玻璃体血管的存活和稳定。我们认为，拟议的研究应该为调节玻璃体血管退行性变的细胞和分子相互作用提供新的见解。？A3/A1-晶体蛋白可能在玻璃体血管退行性变中发挥作用的可能性是重要的；它可能有助于阐明潜在的临床意义的PFV的机制。 持续性胎儿血管畸形(PFV)是一种常见的潜在致盲的先天性眼病。我们的数据(已发表和未发表)表明？A3/A1-晶体蛋白在PFV中起作用。拟议的研究可能有助于阐明导致PFV的机制，并具有潜在的治疗意义。

DESCRIPTION (provided by applicant): This application is in response to the recently announced availability of Recovery Act Funds for Competitive Revision Applications (NOT-OD-09-058). It will create two new jobs in our laboratory and help to retain several jobs at the Johns Hopkins University School of Medicine ES Cell Targeting Core Laboratory, as some of the studies proposed in this revised application will be done at the core facility as a paid service. We previously described a naturally occurring mutation (Nuc1) in the Sprague-Dawley rat with a novel eye phenotype involving cataract, retention of fetal vasculature, and developmental abnormalities in the retina. We have recently reported that the Nuc1 phenotype in which the development of both the lens and retina is abnormal results from mutation of the ?A3/A1-crystallin gene. ?A3/A1-crystallin is expressed in lens fiber cells; our recent studies show that in neural retina it is expressed only in astrocytes. Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress. It is a common congenital developmental disorder of the eye found in an otherwise normal child. The underlying cause of PFV disease is not well understood. The Nuc1 spontaneous mutant rat is the only model that accurately resembles all the clinical symptoms of human PFV disease. In the present competitive supplement, we propose to further characterize our recently established ?A3/A1-crystallin transgenic mouse lines and to generate conditional knockout mice where ?A3/A1-crystallin will be selectively deleted in astrocytes, or the lens. These genetically engineered mouse lines will provide additional tools for gain and loss-of-function studies and will generate more definitive data on the effects of eliminating or modulating the expression of ?A3/A1-crystallin, in the etiology of PFV disease. The studies proposed in this revision application are based on progress made to date under the parent grant and are specifically designed to augment the experiments outlined in the Specific Aims of that grant. Our working hypothesis is that "mutation of ?A3/A1- crystallin causes abnormal association of astrocytes with the hyaloid artery, which inhibits regression of the fetal vasculature". To test this hypothesis, the following specific aims were proposed in the parent grant: SPECIFIC AIM 1: To characterize and compare ?A3/A1-crystallin expression in wildtype and in Nuc1 homozygous rats during lens fiber cell and astrocyte development. SPECIFIC AIM 2: To investigate if altered motility of Nuc1 homozygous astrocytes during development contributes to the abnormal association between astrocytes and the hyaloid vasculature. SPECIFIC AIM 3: To determine if VEGF produced by astrocytes expressing mutant ?A3/A1-crystallin mediates the survival and stabilization of the hyaloid vasculature in the Nuc1 rat. We believe that the proposed studies should provide new insights into the cellular and molecular interactions that regulate hyaloid vascular regression. The possibility that ?A3/A1-crystallin may have a role in hyaloid vascular regression is important; it may help elucidate mechanisms underlying PFV that would have potential clinical implications. PUBLIC HEALTH RELEVANCE Persistent Fetal Vasculature (PFV) is a common potentially blinding, congenital eye disease. Our data (published and unpublished) indicate that ?A3/A1- crystallin plays a role in PFV. The proposed studies may help elucidate mechanisms that lead to PFV and would have potential therapeutic implications.